Project Summary/Abstract The advent of the Zika virus (ZIKV) epidemic around the world has and will cause problems for the correct diagnosis of flavivirus infections. For many years, DENV serological diagnosis was relatively straightforward. However, after the recent ZIKV epidemic, cross-reactive antibodies (Abs) generated after ZIKV infection will bind to DENV even if the patient has never had DENV. The only FDA approved method to differentiate infection by these related viruses is to carry out the Plaque Reduction Neutralization Test (PRNT). This assay is laborious, expensive, not easily adapted to clinical laboratories, and the results are often equivocal. We have already faced this problem with diagnosis of new ZIKV infections at our company Z-Quick, LLC. At Z-Quick, we have already solved the inability to distinguish between ZIKV- and DENV-exposure by using a monoclonal Ab (mAb) competition-based assay. We plan, therefore, to isolate broadly reactive DENV-specific mAbs and develop a similar highly specific, mAb competition-based approach for the diagnosis of DENV. We have isolated over 300 anti-DENV human mAbs from DENV-exposed patients and DENV vaccinees. We will screen these mAbs at the University of Miami for binding properties, specifically, mAbs that recognize DENV and not ZIKV. We have previously developed a rapid and innovative mAb competition-based assay to detect previous ZIKV exposure that can discriminate between DENV and ZIKV with extraordinary specificity. If we are successful, we will: (1) Establish a robust, rapid, and highly specific test for prior DENV exposure that is not complicated by prior ZIKV infection; (2) test our diagnostic assay on complex, well- defined human clinical samples so that our assay can be offered immediately as a laboratory developed test (LDT). !